Tobacco smoke triggers multiple responses in the lung, including mucous and squamous metaplasia. Because squamous, but not mucous metaplasia can be pre-neoplastic, it is important to understand how cells commit themselves to one outcome or the other. Mucous and squamous lesions can be studied mechanistically by monitoring expression of the mucous gene MUC 5 AC and the squamous gene SPRRI. In specific aim 1, based on preliminary data showing that mutagenesis of both TRE and RARE sites diminish MUC 5ACs transcriptional response to smoke, we will transfect lung epithelial cells with luciferase constructs driven by concatamers of the following sites: TRE, RARE and TRE-RARE combined. To determine the intrinsic transactivation potential of putative binding proteins, we will overexpress/delete common factors known to bind each site. We will perform super shift and ChIP assays to identify relevant protein binding in cells in which TRE- and RARE-binding proteins have been experimentally manipulated. In specific aim 2, based on data showing that smoke-exposed rats develop squamous metaplasia in the nose and mucous metaplasia in the airways, we will dissect nasal and airway tissues from smoke- vs. air-exposed rats and perform laser capture microdissection, TaqMan PCR, super shift and ChIP assays to identify TRE and RARE-binding proteins upregulated in smoke-induced squamous vs. mucous cells. In specific aim 3, we will examine squamous and mucous lesions in the lungs of human smokers, using laser capture microdissection followed by TaqMan PCR to quantify expression of relevant transcription factors. We will also retrovirally infect primary human airway cells obtained at autopsy with MUC 5AC-Ds Red 2 and SPRR1-E-YFP prior to exposing them to smoke. Because of the documented heterogeneity of primary cells obtained in this way, we expect some cells to express one fluorophore and others to express the other. We will FACS sort these cells and assay them using TaqMan PCR, ChIP and gel super shift, to determine which TRE and RARE transcription factors are being produced and used in response to smoke by cells exposing MUC 5 AC vs. SPRRI. Finally, we will test the hypothesis that the identity of available TRE- and RARE-binding proteins dictates squamous vs. mucous phenotype by overexpressing panels of transcription factors characteristic of squamous and mucous cells in primary airway epithelial cells or a model system such as CHO cells.